Transcriptional profiling of embryo cryotolerance.

The cryosurvival of embryos is a complex process involving dynamic and integrated morphological, functional, and molecular changes. Here, we evaluated the transcriptional profiling of bovine embryos possessing high and low cryotolerance (HC and LC, respectively) by assessing the resumption of development. Embryos were produced in vitro (N = 1137) and cryopreserved (N = 894). Blastocysts samples possessed pronounced group individualization at RNA sequencing. A total of 114 genes were differentially expressed, and 27 and 84 genes were upregulated in HC and LC, respectively. Among the over-represented biological functions, cellular growth and proliferation, cell death and survival, and organismal survival were predicted to be activated, while cellular movement and cell-to-cell signaling were predicted to be inhibited in HC embryos. Enriched canonical pathways and upstream regulators related to cellular proliferation and survival (HC), inflammatory processes, and cell death (LC) were predicted to represent two embryonic molecular profiles present during the resumption of development after cryopreservation. The marked contrast in transcriptional profiles between HC and LC strongly suggests the influence of embryonic competence after cryopreservation on its respective transcriptome and indicated that HC and LC presented two different molecular strategies to overcome cryopreservation-related stress and resume postcryopreservation development.

Influence of follicle size on bovine oocyte lipid composition, follicular metabolic and stress markers, embryo development and blastocyst lipid content.

This study assessed the lipid composition of oocytes from different follicle sizes and compared the expression of lipid-related genes and follicular fluid (FF) molecules between groups. We also investigated the functional consequences of differences on embryo development and blastocyst lipid deposits. Oocytes and FF were recovered from different follicle sizes. Oocytes from small (≤5mm) and large (≥6mm) bovine follicles were used to produce Day 7 expanded blastocysts (Day7Ex) and blastocysts that only became expanded at Day 8 (Day8Ex) after insemination. Oocytes from >8mm follicles had the highest lipid content. Few oocyte phospholipid variations were identified between groups. Very long chain fatty acid elongase 6 (ELOVL6) mRNA abundance was reduced in larger follicle-derived oocytes compared with the ≤2mm group. Increased levels of glucose, reactive oxygen species, glutathione and superoxide dismutase activity were also identified in FF from larger follicles. Large follicle-derived embryo development and lipid content of Day7Ex were greater than those derived from small follicles. Day8Ex had greater lipid deposition than Day7Ex. Oocytes and blastocysts exhibited follicle size-specific lipids. Large-follicle oocytes had increased lipid content and became Day7Ex with greater lipid deposition whereas delayed blastocoel expansion associated with a prolonged period of culture determined the lipid accumulation of Day8Ex. The FF microenvironment of large follicles seems to favour embryo development.

Lipidome signatures in early bovine embryo development.

Mammalian preimplantation embryonic development is a complex, conserved, and well-orchestrated process involving dynamic molecular and structural changes. Understanding membrane lipid profile fluctuation during this crucial period is fundamental to address mechanisms governing embryogenesis. Therefore, the aim of the present work was to perform a comprehensive assessment of stage-specific lipid profiles during early bovine embryonic development and associate with the mRNA abundance of lipid metabolism-related genes (ACSL3, ELOVL5, and ELOVL6) and with the amount of cytoplasmic lipid droplets. Immature oocytes were recovered from slaughterhouse-derived ovaries, two-cell embryos, and eight- to 16-cell embryos, morula, and blastocysts that were in vitro produced under different environmental conditions. Lipid droplets content and mRNA transcript levels for ACSL3, ELOVL5, and ELOVL6, monitored by lipid staining and quantitative polymerase chain reaction, respectively, increased at morula followed by a decrease at blastocyst stage. Relative mRNA abundance changes of ACSL3 were closely related to cytoplasmic lipid droplet accumulation. Characteristic dynamic changes of phospholipid profiles were observed during early embryo development and related to unsaturation level, acyl chain length, and class composition. ELOVL5 and ELOVL6 mRNA levels were suggestive of overexpression of membrane phospholipids containing elongated fatty acids with 16, 18, and 20 carbons. In addition, putative biomarkers of key events of embryogenesis, embryo lipid accumulation, and elongation were identified. This study provides a comprehensive description of stage-specific lipidome signatures and proposes a mechanism to explain its potential relationship with the fluctuation of both cytoplasmic lipid droplets content and mRNA levels of lipid metabolism-related genes during early bovine embryo development.

The periovulatory endocrine milieu affects the uterine redox environment in beef cows.

BACKGROUND: In cattle, recent studies have shown positive associations between pre-ovulatory concentrations of estradiol (E2), progesterone (P4) at early diestrus and fertility. However, information on cellular and molecular mechanisms through which sex steroids regulate uterine function to support early pregnancy is lacking. Based on endometrial transcriptome data, objective was to compare function of the redox system in the bovine uterus in response to different periovulatory endocrine milieus. METHODS: We employed an animal model to control growth of the pre-ovulatory follicle and subsequent corpus luteum (CL). The large follicle-large CL group (LF-LCL, N=42) presented greater levels of E2 on the day of GnRH treatment (D0; 2.94 vs. 1.27 pg/mL; P=0.0007) and P4 at slaughter on D7 (3.71 vs. 2.62 ng/mL, P=0.01), compared with the small follicle-small CL group (SF-SCL, N=41). Endometrium and uterine washings (N=9, per group) were collected for analyses of variables associated with the uterine redox system. RESULTS: The SF-SCL group had lower endometrial catalase (0.5 vs. 0.79 U/mg protein, P<0.001) and glutathione peroxidase (GPx; 2.0 vs. 2.43 nmol ß-nicotinamide adenine dinucleotide phosphate reduced/min/mg protein, P=0.04) activity, as well as higher lipid peroxidation (28.5 vs. 17.43 nmol malondialdehyde/mg of protein, P<0.001) and superoxide dismutase (SOD) activity (44.77 vs. 37.76 U; P=0.04). There were no differences in the endometrial reactive species (RS) or glutathione (GSH) concentrations between the groups. The uterine washing samples showed no differences in the concentrations of RS or GSH or in total SOD activity (P>0.1). Additionally, catalase, GPx4, SOD1 and SOD2 gene expression was lower in the SF-SCL group than in the LF-LCL group. CONCLUSIONS: We concluded that the intrauterine environment of cows from the LF-LCL group exhibited higher antioxidant activity than that of the cows from the SF-SCL group. We speculate that uterine receptivity and fertility are associated with an optimal redox environment, such as that present in the animals in the LF-LCL group.

Supplementation with sunflower seed increases circulating cholesterol concentrations and potentially impacts on the pregnancy rates in Bos indicus beef cattle.

We aimed to evaluate the effect of supplementation with sunflower seed on blood concentrations of progesterone and cholesterol and on the pregnancy rate in beef cattle subjected to timed artificial insemination (TAI) and timed embryo transfer (TET). In experiment 1, cows were received 22-day supplements containing (sunflower, n = 66) sunflower seed or not (control, n = 67) immediately after a progesterone/estradiol-based TAI protocol (Day 0). The cholesterol concentration on Day 21 and the pregnancy rate were greater (P < 0.03) in the sunflower group (148.2 ± 6.1 mg/dL and 66.7%) than those in the control group (116.0 ± 6.4 mg/dL and 47.8%). In experiment 2, heifers received an in vitro-produced embryo 7 days after the expected time of the synchronized ovulation. Heifers were separated into two supplementation groups (sunflower, n = 106 and control, n = 111) for 22 days. The plasma progesterone concentration on Day 7 was not different between the groups. However, on Day 19, the plasma progesterone concentration was greater (P < 0.0001) in the sunflower group (5.8 ± 0.4 ng/mL) than that in the control group (3.5 ± 0.4 ng/mL). A greater (P < 0.05) cholesterol concentration was observed in the sunflower group than that in the control group on Days 7 (306.0 ± 11.6 vs. 277.1 ± 11.9 mg/dL, respectively) and 19 (260.5 ± 8.0 vs. 232.0 ± 8.0 mg/dL, respectively). The pregnancy rate was greater (P = 0.01) in the sunflower-treated heifers (55.7%) than that in control-treated heifers (36.9%). Results indicate that sunflower seed supplementation increases the circulating cholesterol concentrations and potentially impacts the pregnancy rate in suckled beef cattle subjected to TAI or TET.

Spatio-specific regulation of endocrine-responsive gene transcription by periovulatory endocrine profiles in the bovine reproductive tract.

In cattle, pro-oestrous oestradiol and dioestrous progesterone concentrations modulate endometrial gene expression and fertility. The aim was to compare the effects of different periovulatory endocrine profiles on the expression of progesterone receptor (PGR), oestrogen receptor 2 (ESR2), oxytocin receptor (OXTR), member C4 of aldo-keto reductase family 1 (AKR1C4), lipoprotein lipase (LPL), solute carrier family 2, member 1 (SLC2A1) and serpin peptidase inhibitor, clade A member 14 (SERPINA14): (1) between uterine horns ipsi- and contralateral to the corpus luteum (CL), (2) between regions of the ipsilateral horn and (3) in the vagina. Endometrium and vagina tissue samples were collected from cows that ovulated a larger (large follicle-large CL, LF-LCL; n=6) or smaller follicle (small follicle-small CL, SF-SCL; n=6) 7 days after oestrus. Cows in the LF-LCL group had a greater abundance of transcripts encoding ESR2, AKR1C4, LPL, SLC2A1 and SERPINA14, but a reduced expression of PGR and OXTR in the endometrium versus the SF-SCL group (PPGR and OXTR was greater in the contralateral compared with the ipsilateral horn (PPGR, ESR2, LPL, SLC2A1 and SERPINA14 (P<0.05). Different periovulatory endocrine profiles, i.e. LF-LCL or SF-SCL, did not influence gene expression in the vagina and had no interaction with inter- or intra-uterine horn gene expression. In conclusion, inter- and intra-uterine horn variations in gene expression indicate that the expression of specific genes in the bovine reproductive tract is location dependent. However, spatial distribution of transcripts was not influenced by distinct periovulatory sex-steroid environments.

Influence of cloning by chromatin transfer on placental gene expression at Day 45 of pregnancy in cattle.

Poor success rates in somatic cell cloning are often attributed to abnormal early embryonic development as well as late abnormal fetal growth and placental development. Although promising results have been reported following chromatin transfer (CT), a novel cloning method that includes the remodeling of the donor nuclei in vitro prior to their transfer into enucleated oocytes, animals cloned by CT show placental abnormalities similar to those observed following conventional nuclear transfer. We hypothesized that the placental gene expression pattern from cloned fetuses was ontologically related to the frequently observed placental phenotype. The aim of the present study was to compare global gene expression by microarray analysis of Day 44-47 cattle placentas derived from CT cloned fetuses with those derived from in vitro fertilization (i.e. control), and confirm the altered mRNA and protein expression of selected molecules by qRT-PCR and immunohistochemistry, respectively. The differentially expressed genes identified in the present study are known to be involved in a range of activities associated with cell adhesion, cell cycle control, intracellular transport and proteolysis. Specifically, an imprinted gene, involved with cell proliferation and placentomegaly in humans (CDKN1C) and a peptidase that serves as a marker for non-invasive trophoblast cells in human placentas (DPP4), had mRNA and protein altered in CT placentas. It was concluded that the altered pattern of gene expression observed in CT samples may contribute to the abnormal placental development phenotypes commonly identified in cloned offspring, and that expression of imprinted as well as trophoblast invasiveness-related genes is altered in cattle cloned by CT.

Reactive oxygen species mediate mitogenic growth factor signaling pathways in human leiomyoma smooth muscle cells.

Uterine leiomyomas are benign uterine tumors characterized by extracellular matrix remodeling, increased collagen deposition, and increased smooth muscle cell (SMC) proliferation. The reactive oxygen species (ROS) producing NADPH oxidase complex has been shown to be involved in the signaling pathways of several growth factors, cytokines, and vasoactive agents that stimulate proliferation of a variety of cell types. Our objective was to test the hypothesis that ROS derived from NADPH oxidase is a necessary component of the MAP kinase mitogenic pathway activated by platelet derived growth factor (PDGF) and epidermal growth factor (EGF) in leiomyoma SMCs (LSMCs). Primary cell cultures of LSMCs were used as our experimental model. Our results showed that stimulation of these cells with PDGF or EGF caused a marked increase in intracellular ROS production and that the NADPH oxidase inhibitor, DPI, blocks ROS production. In addition, inhibition of ROS production by NADPH oxidase inhibitors blocked, in a dose-dependent manner, the EGF- and PDGF-induced increase in [(3)H]thymidine incorporation by LSMCs. Furthermore, an exogenous source of ROS, hydrogen peroxide, was sufficient to stimulate [(3)H]thymidine incorporation in LSMCs but did not affect COL1A2 and COL3A1 mRNA levels. Inhibition of the NADPH oxidase complex decreased PDGF-induced MAPK1/MAPK3 activation, whereas exogenous hydrogen peroxide induced MAPK1/MAPK3 activation. This article is the first report suggesting the presence of the NADPH oxidase system and its importance in mitogenic signaling pathways in LSMCs. The necessity of NADPH oxidase-derived ROS for EGF and PDGF signaling pathways leading to cell proliferation points to another potential therapeutic target for treatment and/or prevention of uterine leiomyomas.

Basigin-2 is a cell surface receptor for soluble basigin ligand.

The metastatic spread of a tumor is dependent upon the ability of the tumor to stimulate surrounding stromal cells to express enzymes required for tissue remodeling. The immunoglobulin superfamily protein basigin (EMMPRIN/CD147) is a cell surface glycoprotein expressed by tumor cells that stimulates matrix metalloproteinase and vascular endothelial growth factor expression in stromal cells. The ability of basigin to stimulate expression of molecules involved in tissue remodeling and angiogenesis makes basigin a potential target for the development of strategies to block metastasis. However, the identity of the cell surface receptor for basigin remains controversial. The goal of this study was to determine the identity of the receptor for basigin. Using a novel recombinant basigin protein (rBSG) corresponding to the extracellular domain of basigin, it was demonstrated that the native, nonglycosylated rBSG protein forms dimers in solution. Furthermore, rBSG binds to the surface of uterine fibroblasts, activates the ERK1/2 signaling pathway, and induces expression of matrix metalloproteinases 1, 2, and 3. Proteins that interact with rBSG were isolated using a biotin label transfer technique and sequenced by matrix-assisted laser desorption ionization tandem mass spectrophotometry. The results demonstrate that rBSG interacts with basigin expressed on the surface of fibroblasts and is subsequently internalized. During internalization, rBSG associates with a novel form of human basigin (basigin-3). It was concluded that cell surface basigin functions as a membrane receptor for soluble basigin and this homophilic interaction is not dependent upon glycosylation of the basigin ligand.